title: Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation.
abstract: Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.
authors.affiliation: Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Rasko
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Worsham
authors.forename: Terry G
authors.initials: TG
authors.lastname: Abshire
authors.forename: Scott T
authors.initials: ST
authors.lastname: Stanley
authors.forename: Jason D
authors.initials: JD
authors.lastname: Bannan
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wilson
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Langham
authors.forename: R Scott
authors.initials: RS
authors.lastname: Decker
authors.forename: Lingxia
authors.initials: L
authors.lastname: Jiang
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Read
authors.forename: Adam M
authors.initials: AM
authors.lastname: Phillippy
authors.forename: Steven L
authors.initials: SL
authors.lastname: Salzberg
authors.forename: Mihai
authors.initials: M
authors.lastname: Pop
authors.forename: Matthew N
authors.initials: MN
authors.lastname: Van Ert
authors.forename: Leo J
authors.initials: LJ
authors.lastname: Kenefic
authors.forename: Paul S
authors.initials: PS
authors.lastname: Keim
authors.forename: Claire M
authors.initials: CM
authors.lastname: Fraser-Liggett
authors.forename: Jacques
authors.initials: J
authors.lastname: Ravel
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics*; D023482:Bioterrorism; D004252:DNA Mutational Analysis / Q000379:methods; D044707:Forensic Sciences / Q000379:methods*; D056426:Genetic Loci; D016680:Genome, Bacterial / Q000235:genetics*; D055106:Genome-Wide Association Study / Q000379:methods; D006801:Humans; D009154:Mutation